Paraoxonase

ABSTRACT

Human paraoxonase enzyme and DNA (RNA) encoding such paraoxonase enzymes are disclosed. Also provided is the procedure for producing such polypeptides by recombinant techniques. Uses of such polypeptides include their use as an antidote for organophosphate poisoning and to prevent neuronal cell death.

This invention relates to newly identified polynucleotides, polypeptidesencoded by such polynucleotides, the use of such polynucleotides andpolypeptides, as well as the production of such polynucleotides andpolypeptides. More particularly, the polypeptide of the presentinvention is paraoxonase. The invention also relates to inhibiting theaction of such polypeptides.

Parathion (diethyl-para-nitrophenyl phosphothioate) and chlorpyrifos (O,O-diethyl-O-3, 5, 6-trichloro-2-pyridinol), are commonly usedorganophosphorous insecticides and are involved in a large number ofpoisonings of agricultural workers and others each year (Hayes, W. J.,Pesticides Studied in Man, Wilkins and Wilkins, Baltimore, pp. 284-435(1982)). Both compounds are bioactivated in vivo to form the respectivetoxic oxon inhibitors of cholinesterase. This leads to neuronal celldeath and related neuronal disorders. Both oxons are hydrolyzed by theserum enzyme paraoxonase/arylesterase, most, if not all, of which islocated in the high-density lipoprotein (HDL) particles (Mackness, M.I., et al., Biochem. Pharmacol., 32:2291-2296 (1983)).

In humans, this enzyme exhibits a substrate dependent activitypolymorphism (Mallinckrodt, M. G. and Diepgen, T. L., Toxicol. Environ.Chem., 18:79-196 (1988)). Human serum paraoxonase/arylesterase catalyzesthe hydrolysis of organophosphates, aromatic carboxylic acid esters, andcarbamates. There appears to be an existence of two alleles. One allelicproduct hydrolyses paraoxon with a high turnover number and the otherwith a low turnover number. Other substrates such as phenylacetate, betaand naphthylacetate (Gan, K. N., et al., Drug Metab. Dispos., 19:100-106(1991)) and chlorpyrifos oxon (Furlong, C. E., et al., Anal. Biochem.,180:242-247 (1989)) are hydrolyzed by either allelic product at the sameor nearly the same rate. The enzyme also hydrolyses the nerve agentssoman and sarin (Gan, K. N., et al., Drug Metab. Dispos., 19:100-106(1991)). The hydrolysis of neurotoxic organophosphates is a beneficial,fortuitous activity of paraoxonase.

In accordance with one aspect of the present invention, there isprovided a novel putative mature polypeptide which is paraoxonase, aswell as fragments, analogs and derivatives thereof. The polypeptide ofthe present invention is of human origin.

In accordance with another aspect of the present invention, there areprovided polynucleotides (DNA or RNA) which encode such polypeptides.

In accordance with yet a further aspect of the present invention, thereis provided a process for producing such polypeptides by recombinanttechniques.

In accordance with yet a further aspect of the present invention, thereis provided a process for utilizing such polypeptide, or polynucleotideencoding such polypeptide for therapeutic purposes, for example, as anantidote for organophosphate toxicity (pesticide poisoning) and inpreventing neuronal cell death.

In accordance with yet a further aspect of the present invention, thereare provided antibodies against such polypeptides.

These and other aspects of the present invention should be apparent tothose skilled in the art from the teachings herein.

The following drawings are illustrative of embodiments of the inventionand are not meant to limit the scope of the invention as encompassed bythe claims.

FIG. 1A (sheet 1/2) and FIG. 1B (sheet 2/2), wherein FIG. 1B is acontinuation of the sequence information shown in FIG. 1A, collectivelyshow the cDNA sequence (SEQ ID NO:1) and deduced amino acid sequence(SEQ ID NO:2) for the putative mature paraoxonase polypeptide. Thestandard one-letter abbreviations for amino acids are utilized.

In accordance with an aspect of the present invention, there is providedan isolated nucleic acid (polynucleotide) which encodes for the maturepolypeptide having the deduced amino acid sequence of FIG. 1 or for themature polypeptide encoded by the cDNA of the clone deposited as ATCCDeposit No. 75773 on May 12, 1994.

The polynucleotide of this invention was discovered in a cDNA libraryderived from a human amygdala. It is structurally related to the humanserum paraoxonase/arylesterase family. It contains an open reading frameencoding a protein of approximately 356 amino acid residues. The proteinexhibits the highest degree of homology to serum paraoxonase oforyctolagus cuniculus with 67% identity and 83% similarity over a 249amino acid stretch.

The polynucleotide of the present invention may be in the form of RNA orin the form of DNA, which DNA includes cDNA, genomic DNA, and syntheticDNA. The DNA may be double-stranded or single-stranded, and if singlestranded may be the coding strand or non-coding (anti-sense) strand. Thecoding sequence which encodes the mature polypeptide may be identical tothe coding sequence shown in FIG. 1 or that of the deposited clone ormay be a different coding sequence which coding sequence, as a result ofthe redundancy or degeneracy of the genetic code, encodes the samemature polypeptide as the DNA of FIG. 1 or the deposited cDNA.

The polynucleotide which encodes for the mature polypeptide of FIG. 1 orfor the mature polypeptide encoded by the deposited cDNA may include:only the coding sequence for the mature polypeptide; the coding sequencefor the mature polypeptide and additional coding sequence such as aleader or secretory sequence or a proprotein sequence; the codingsequence for the mature polypeptide (and optionally additional codingsequence) and non-coding sequence, such as introns or non-codingsequence 5' and/or 3' of the coding sequence for the mature polypeptide.

Thus, the term "polynucleotide encoding a polypeptide" encompasses apolynucleotide which includes only coding sequence for the polypeptideas well as a polynucleotide which includes additional coding and/ornon-coding sequence.

The present invention further relates to variants of the hereinabovedescribed polynucleotides which encode for fragments, analogs andderivatives of the polypeptide having the deduced amino acid sequence ofFIG. 1 or the polypeptide encoded by the cDNA of the deposited clone.The variant of the polynucleotide may be a naturally occurring allelicvariant of the polynucleotide or a non-naturally occurring variant ofthe polynucleotide.

Thus, the present invention includes polynucleotides encoding the samemature polypeptide as shown in FIG. 1 or the same mature polypeptideencoded by the cDNA of the deposited clone as well as variants of suchpolynucleotides which variants encode for a fragment, derivative oranalog of the polypeptide of FIG. 1 or the polypeptide encoded by thecDNA of the deposited clone. Such nucleotide variants include deletionvariants, substitution variants and addition or insertion variants.

As hereinabove indicated, the polynucleotide may have a coding sequencewhich is a naturally occurring allelic variant of the coding sequenceshown in FIG. 1 or of the coding sequence of the deposited clone. Asknown in the art, an allelic variant is an alternate form of apolynucleotide sequence which may have a substitution, deletion oraddition of one or more nucleotides, which does not substantially alterthe function of the encoded polypeptide.

The present invention also includes polynucleotides, wherein the codingsequence for the mature polypeptide may be fused in the same readingframe to a polynucleotide sequence which aids in expression andsecretion of a polypeptide from a host cell, for example, a leadersequence which functions as a secretory sequence for controllingtransport of a polypeptide from the cell. The polypeptide having aleader sequence is a preprotein and may have the leader sequence cleavedby the host cell to form the mature form of the polypeptide. Thepolynucleotides may also encode for a proprotein which is the matureprotein plus additional 5' amino acid residues. A mature protein havinga prosequence is a proprotein and is an inactive form of the protein.Once the prosequence is cleaved an active mature protein remains.

Thus, for example, the polynucleotide of the present invention mayencode for a mature protein, or for a protein having a prosequence orfor a protein having both a prosequence and a presequence (leadersequence).

The polynucleotides of the present invention may also have the codingsequence fused in frame to a marker sequence which allows forpurification of the polypeptide of the present invention. The markersequence may be a hexa-histidine tag supplied by a pQE-9 vector toprovide for purification of the mature polypeptide fused to the markerin the case of a bacterial host, or, for example, the marker sequencemay be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells,is used. The HA tag corresponds to an epitope derived from the influenzahemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)).

The present invention further relates to polynucleotides which hybridizeto the hereinabove-described sequences if there is at least 50% andpreferably 70% identity between the sequences. The present inventionparticularly relates to polynucleotides which hybridize under stringentconditions to the hereinabove-described polynucleotides. As herein used,the term "stringent conditions" means hybridization will occur only ifthere is at least 95% and preferably at least 97% identity between thesequences. The polynucleotides which hybridize to the hereinabovedescribed polynucleotides in a preferred embodiment encode polypeptideswhich retain substantially the same biological function or activity asthe mature polypeptide encoded by the cDNA of FIG. 1 or the depositedcDNA.

The deposit(s) referred to herein will be maintained under the terms ofthe Budapest Treaty on the International Recognition of the Deposit ofMicro-organisms for purposes of Patent Procedure. These deposits areprovided merely as convenience to those of skill in the art and are notan admission that a deposit is required under 35 U.S.C. §112. Thesequence of the polynucleotides contained in the deposited materials, aswell as the amino acid sequence of the polypeptides encoded thereby, areincorporated herein by reference and are controlling in the event of anyconflict with any description of sequences herein. A license may berequired to make, use or sell the deposited materials, and no suchlicense is hereby granted.

The present invention further relates to a paraoxonase polypeptide whichhas the deduced amino acid sequence of FIG. 1 or which has the aminoacid sequence encoded by the deposited cDNA, as well as fragments,analogs and derivatives of such polypeptide.

The terms "fragment," "derivative" and "analog" when referring to thepolypeptide of FIG. 1 or that encoded by the deposited cDNA, means apolypeptide which retains essentially the same biological function oractivity as such polypeptide. Thus, an analog includes a proproteinwhich can be activated by cleavage of the proprotein portion to producean active mature polypeptide.

The polypeptide of the present invention may be a recombinantpolypeptide, a natural polypeptide or a synthetic polypeptide,preferably a recombinant polypeptide.

The fragment, derivative or analog of the polypeptide of FIG. 1 or thatencoded by the deposited cDNA may be (i) one in which one or more of theamino acid residues are substituted with a conserved or non-conservedamino acid residue (preferably a conserved amino acid residue) and suchsubstituted amino acid residue may or may not be one encoded by thegenetic code, or (ii) one in which one or more of the amino acidresidues includes a substituent group, or (iii) one in which the maturepolypeptide is fused with another compound, such as a compound toincrease the half-life of the polypeptide (for example, polyethyleneglycol), or (iv) one in which the additional amino acids are fused tothe mature polypeptide, such as a leader or secretory sequence or asequence which is employed for purification of the mature polypeptide ora proprotein sequence. Such fragments, derivatives and analogs aredeemed to be within the scope of those skilled in the art from theteachings herein.

The polypeptides and polynucleotides of the present invention arepreferably provided in an isolated form, and preferably are purified tohomogeneity.

The term "isolated" means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

The present invention also relates to vectors which includepolynucleotides of the present invention, host cells which aregenetically engineered with vectors of the invention and the productionof polypeptides of the invention by recombinant techniques.

Host cells are genetically engineered (transduced or transformed ortransfected) with the vectors of this invention which may be, forexample, a cloning vector or an expression vector. The vector may be,for example, in the form of a plasmid, a viral particle, a phage, etc.The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants or amplifying the serum paraoxonase genes. The cultureconditions, such as temperature, pH and the like, are those previouslyused with the host cell selected for expression, and will be apparent tothe ordinarily skilled artisan.

The polynucleotides of the present invention may be employed forproducing polypeptides by recombinant techniques. Thus, for example, thepolynucleotide may be included in any one of a variety of expressionvectors for expressing a polypeptide. Such vectors include chromosomal,nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40;bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectorsderived from combinations of plasmids and phage DNA, viral DNA such asvaccinia, adenovirus, fowl pox virus, and pseudorabies. However, anyother vector may be used as long as it is replicable and viable in thehost.

The appropriate DNA sequence may be inserted into the vector by avariety of procedures. In general, the DNA sequence is inserted into anappropriate restriction endonuclease site(s) by procedures known in theart. Such procedures and others are deemed to be within the scope ofthose skilled in the art.

The DNA sequence in the expression vector is operatively linked to anappropriate expression control sequence(s) (promoter) to direct mRNAsynthesis. As representative examples of such promoters, there may bementioned: LTR or SV40 promoter, the E. coli. lac or trp, the phagelambda P_(L) promoter and other promoters known to control expression ofgenes in prokaryotic or eukaryotic cells or their viruses. Theexpression vector also contains a ribosome binding site for translationinitiation and a transcription terminator. The vector may also includeappropriate sequences for amplifying expression.

In addition, the expression vectors preferably contain one or moreselectable marker genes to provide a phenotypic trait for selection oftransformed host cells such as dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, or such as tetracycline orampicillin resistance in E. coli.

The vector containing the appropriate DNA sequence as hereinabovedescribed, as well as an appropriate promoter or control sequence, maybe employed to transform an appropriate host to permit the host toexpress the protein.

As representative examples of appropriate hosts, there may be mentioned:bacterial cells, such as E. coli, Streptomyces, Salmonella typhimurium;fungal cells, such as yeast; insect cells such as Drosophila and Sf9;animal cells such as CHO, COS or Bowes melanoma; plant cells, etc. Theselection of an appropriate host is deemed to be within the scope ofthose skilled in the art from the teachings herein.

More particularly, the present invention also includes recombinantconstructs comprising one or more of the sequences as broadly describedabove. The constructs comprise a vector, such as a plasmid or viralvector, into which a sequence of the invention has been inserted, in aforward or reverse orientation. In a preferred aspect of thisembodiment, the construct further comprises regulatory sequences,including, for example, a promoter, operably linked to the sequence.Large numbers of suitable vectors and promoters are known to those ofskill in the art, and are commercially available. The following vectorsare provided by way of example. Bacterial: pQE70, pQE60, pQE-9 (Qiagen),pbs, pD10, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a,pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5(Pharmacia). Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene)pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other plasmid orvector may be used as long as they are replicable and viable in thehost.

Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are PKK232-8 and PCM7. Particular namedbacterial promoters include lacI, lacZ, T3, T7, gpt, lambda P_(R), P_(L)and trp. Eukaryotic promoters include CMV immediate early, HSV thymidinekinase, early and late SV40, LTRs from retrovirus, and mousemetallothionein-I. Selection of the appropriate vector and promoter iswell within the level of ordinary skill in the art.

In a further embodiment, the present invention relates to host cellscontaining the above-described constructs. The host cell can be a highereukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell,such as a yeast cell, or the host cell can be a prokaryotic cell, suchas a bacterial cell. Introduction of the construct into the host cellcan be effected by calcium phosphate transfection, DEAE-Dextran mediatedtransfection, or electroporation. (Davis, L., Dibner, M., Battey, I.,Basic Methods in Molecular Biology, (1986)).

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence.Alternatively, the polypeptides of the invention can be syntheticallyproduced by conventional peptide synthesizers.

Mature proteins can be expressed in mammalian cells, yeast, bacteria, orother cells under the control of appropriate promoters. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the present invention.Appropriate cloning and expression vectors for use with prokaryotic andeukaryotic hosts are described by Sambrook, et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), thedisclosure of which is hereby incorporated by reference.

Transcription of the DNA encoding the polypeptides of the presentinvention by higher eukaryotes is increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp that act on a promoter to increase itstranscription. Examples including the SV40 enhancer on the late side ofthe replication origin bp 100 to 270, a cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTRP1 gene, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Such promoters can bederived from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK), α-factor, acid phosphatase, or heatshock proteins, among others. The heterologous structural sequence isassembled in appropriate phase with translation initiation andtermination sequences, and preferably, a leader sequence capable ofdirecting secretion of translated protein into the periplasmic space orextracellular medium. Optionally, the heterologous sequence can encode afusion protein including an N-terminal identification peptide impartingdesired characteristics, e.g., stabilization or simplified purificationof expressed recombinant product.

useful expression vectors for bacterial use are constructed by insertinga structural DNA sequence encoding a desired protein together withsuitable translation initiation and termination signals in operablereading phase with a functional promoter. The vector will comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector and to, if desirable, provideamplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus, although others may also be employedas a matter of choice.

As a representative but nonlimiting example, useful expression vectorsfor bacterial use can comprise a selectable marker and bacterial originof replication derived from commercially available plasmids comprisinggenetic elements of the well known cloning vector pBR322 (ATCC 37017).Such commercial vectors include, for example, pKK223-3 (Pharmacia FineChemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, Wis.,USA). These pBR322 "backbone" sections are combined with an appropriatepromoter and the structural sequence to be expressed.

Following transformation of a suitable host strain and growth of thehost strain to an appropriate cell density, the selected promoter isinduced by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period.

Cells are typically harvested by centrifugation, disrupted by physicalor chemical means, and the resulting crude extract retained for furtherpurification.

Microbial cells employed in expression of proteins can be disrupted byany convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents, such methods arewell know to those skilled in the art.

Various mammalian cell culture systems can also be employed to expressrecombinant protein. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblasts, described by Gluzman,Cell, 23:175 (1981), and other cell lines capable of expressing acompatible vector, for example, the C127, 3T3, CHO, HeLa and BHK celllines. Mammalian expression vectors will comprise an origin ofreplication, a suitable promoter and enhancer, and also any necessaryribosome binding sites, polyadenylation site, splice donor and acceptorsites, transcriptional termination sequences, and 5' flankingnontranscribed sequences. DNA sequences derived from the SV40 splice,and polyadenylation sites may be used to provide the requirednontranscribed genetic elements.

The paraoxonase polypeptides can be recovered and purified fromrecombinant cell cultures by methods including ammonium sulfate orethanol precipitation, acid extraction, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography hydroxylapatite chromatographyand lectin chromatography. Protein refolding steps can be used, asnecessary, in completing configuration of the mature protein. Finally,high performance liquid chromatography (HPLC) can be employed for finalpurification steps.

The polypeptides of the present invention may be a naturally purifiedproduct, or a product of chemical synthetic procedures, or produced byrecombinant techniques from a prokaryotic or eukaryotic host (forexample, by bacterial, yeast, higher plant, insect and mammalian cellsin culture). Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated. Polypeptides of the inventionmay also include an initial methionine amino acid residue.

The paraoxonase polypeptides of the present invention are useful as anantidote for organophosphate poisoning, since the toxic oxon inhibitorsof cholinesterase are hydrolyzed by paraoxonase.

The paraoxonase polypeptides are also useful for preventing neuronalcell death due to such toxic poisoning. If organophosphate poisoning isleft untreated, neuronal cell death will result.

The polypeptide of the present invention is also useful for identifyingother molecules which have similar biological activity. An example of ascreen for this is isolating the coding region of the paraoxonase geneby using the known DNA sequence to synthesize an oligonucleotide probe.Labeled oligonucleotides having a sequence complementary to that of thegene of the present invention are used to screen a library of humancDNA, genomic DNA or mRNA to determine which members of the library theprobe hybridizes to.

The polypeptides may also be employed in accordance with the presentinvention by expression of such polypeptides in vivo, which is oftenreferred to as "gene therapy."

Thus, for example, cells from a patient may be engineered with apolynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with theengineered cells then being provided to a patient to be treated with thepolypeptide. Such methods are well-known in the art. For example, cellsmay be engineered by procedures known in the art by use of a retroviralparticle containing RNA encoding a polypeptide of the present invention.

Similarly, cells may be engineered in vivo for expression of apolypeptide in vivo by, for example, procedures known in the art. Asknown in the art, a producer cell for producing a retroviral particlecontaining RNA encoding the polypeptide of the present invention may beadministered to a patient for engineering cells in vivo and expressionof the polypeptide in vivo. These and other methods for administering apolypeptide of the present invention by such method should be apparentto those skilled in the art from the teachings of the present invention.For example, the expression vehicle for engineering cells may be otherthan a retrovirus, for example, an adenovirus which may be used toengineer cells in vivo after combination with a suitable deliveryvehicle.

The polypeptides of the present invention may be employed in combinationwith a suitable pharmaceutical carrier. Such compositions comprise atherapeutically effective amount of the polypeptide, and apharmaceutically acceptable carrier or excipient. Such a carrierincludes but is not limited to saline, buffered saline, dextrose, water,glycerol, ethanol, and combinations thereof. The formulation should suitthe mode of administration.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration. In addition, thepolypeptides of the present invention may be employed in conjunctionwith other therapeutic compounds.

The pharmaceutical compositions may be administered in a convenientmanner such as by the oral, topical, intravenous, intraperitoneal,intramuscular, subcutaneous, intranasal or intradermal routes.Paraoxonase is administered in an amount which is effective for treatingand/or prophylaxis of the specific indication. In general, paraoxonasewill be administered in an amount of at least about 10 μg/kg body weightand in most cases they will be administered in an amount not in excessof about 8 mg/Kg body weight per day. In most cases, the dosage is fromabout 10 μg/kg to about 1 mg/kg body weight daily, taking into accountthe routes of administration, symptoms, etc.

The sequences of the present invention are also valuable for chromosomeidentification. The sequence is specifically targeted to and canhybridize with a particular location on an individual human chromosome.The paraoxonase gene is located close to the cystic fibrosis gene onchromosome 7. Moreover, there is a current need for identifyingparticular sites on the chromosome. Few chromosome marking reagentsbased on actual sequence data (repeat polymorphisms) are presentlyavailable for marking chromosomal location. The mapping of DNAs tochromosomes according to the present invention is an important firststep in correlating those sequences with genes associated with disease.

Briefly, sequences can be mapped to chromosomes by preparing PCR primers(preferably 15-25 bp) from the cDNA. Computer analysis of the cDNA isused to rapidly select primers that do not span more than one exon inthe genomic DNA, thus complicating the amplification process. Theseprimers are then used for PCR screening of somatic cell hybridscontaining individual human chromosomes. Only those hybrids containingthe human gene corresponding to the primer will yield an amplifiedfragment.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning aparticular DNA to a particular chromosome. Using the present inventionwith the same oligonucleotide primers, sublocalization can be achievedwith panels of fragments from specific chromosomes or pools of largegenomic clones in an analogous manner. Other mapping strategies that cansimilarly be used to map to its chromosome include in situhybridization, prescreening with labeled flow-sorted chromosomes andpreselection by hybridization to construct chromosome specific-cDNAlibraries.

Fluorescence in situ hybridization (FISH) of a cDNA clone to a metaphasechromosomal spread can be used to provide a precise chromosomal locationin one step. This technique can be used with cDNA as short as 500 or 600bases; however, clones larger than 2,000 bp have a higher likelihood ofbinding to a unique chromosomal location with sufficient signalintensity for simple detection. FISH requires use of the clones fromwhich the EST was derived, and the longer the better. For example, 2,000bp is good, 4,000 is better, and more than 4,000 is probably notnecessary to get good results a reasonable percentage of the time. For areview of this technique, see Verma et al., Human Chromosomes: a Manualof Basic Techniques, Pergamon Press, New York (1988).

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. Such data are found, for example, in V. McKusick,Mendelian Inheritance in Man (available on line through Johns HopkinsUniversity Welch Medical Library). The relationship between genes anddiseases that have been mapped to the same chromosomal region are thenidentified through linkage analysis (coinheritance of physicallyadjacent genes).

With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of between 50 and 500 potentialcausative genes. (This assumes 1 megabase mapping resolution and onegene per 20 kb).

The polypeptides, their fragments or other derivatives, or analogsthereof, or cells expressing them can be used as an immunogen to produceantibodies thereto. These antibodies can be, for example, polyclonal ormonoclonaI antibodies. The present invention also includes chimeric,single chain, and humanized antibodies, as well as Fab fragments, or theproduct of an Fab expression library. Various procedures known in theart may be used for the production of such antibodies and fragments.

Antibodies generated against the polypeptides corresponding to asequence of the present invention can be obtained by direct injection ofthe polypeptides into an animal or by administering the polypeptides toan animal, preferably a nonhuman. The antibody so obtained will thenbind the polypeptides itself. In this manner, even a sequence encodingonly a fragment of the polypeptides can be used to generate antibodiesbinding the whole native polypeptides. Such antibodies can then be usedto isolate the polypeptide from tissue expressing that polypeptide.

For preparation of monoclonal antibodies, any technique which providesantibodies produced by continuous cell line cultures can be used.Examples include the hybridoma technique (Kohler and Milstein, 1975,Nature, 256:495-497), the trioma technique, the human B-cell hybridomatechnique (Kozbor et al., 1983, Immunology Today 4:72), and theEBV-hybridoma technique to produce human monoclonal antibodies (Cole, etal., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc., pp. 77-96).

Techniques described for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce single chain antibodies toimmunogenic polypeptide products of this invention.

This invention also provides a method of screening drugs to identifythose which enhance (agonists) the interaction of paraoxonase with itssubstrate which comprises, for example, contacting a mammalian cellcomprising a DNA molecule encoding paraoxonase with a plurality of drugsand parathion or chlorpyrifos and detecting those drugs which enhancethe hydrolysis of the toxic oxons by paraoxonase and thereby identifyingdrugs which specifically act as agonists. Various methods of detectionmay be employed. The toxic oxons may be "labeled" by association with adetectable marker substance (e.g., radiolabel or a non-isotopic label,such as biotin) such that their hydrolysis may be measured. Drugcandidates are identified by choosing chemical compounds which bind withhigh affinity to the expressed paraoxonase polypeptide in transfectedcells, using radioligand binding methods well-known in the art.

The present invention will be further described with reference to thefollowing examples; however, it is to be understood that the presentinvention is not limited to such examples. All parts or amounts, unlessotherwise specified, are by weight.

In order to facilitate understanding of the following examples certainfrequently occurring methods and/or terms will be described.

"Plasmids" are designated by a lower case p preceded and/or followed bycapital letters and/or numbers. The starting plasmids herein are eithercommercially available, publicly available on an unrestricted basis, orcan be constructed from available plasmids in accord with publishedprocedures. In addition, equivalent plasmids to those described areknown in the art and will be apparent to the ordinarily skilled artisan.

"Digestion" of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 μg of plasmid or DNA fragment is used with about 2units of enzyme in about 20 μl of buffer solution. For the purpose ofisolating DNA fragments for plasmid construction, typically 5 to 50 μgof DNA are digested with 20 to 250 units of enzyme in a larger volume.Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by the manufacturer. Incubation times of about 1hour at 37° C. are ordinarily used, but may vary in accordance with thesupplier's instructions. After digestion the reaction is electrophoreseddirectly on a polyacrylamide or agarose gel to isolate the desiredfragment.

Size separation of the cleaved fragments is performed using 8 percentpolyacrylamide gel described by Goeddel, D. et al., Nucleic Acids Res.,8:4057 (1980).

"Oligonucleotides" refers to either a single strandedpolydeoxynucleotide or two complementary polydeoxynucleotide strandswhich may be chemically synthesized. Such synthetic oligonucleotideshave no 5' phosphate and thus will not ligate to another oligonucleotidewithout adding a phosphate with an ATP in the presence of a kinase. Asynthetic oligonucleotide will ligate to a fragment that has not beendephosphorylated.

"Ligation" refers to the process of forming phosphodiester bonds betweentwo double stranded nucleic acid fragments (Maniatis, T., et al., Id.,p. 146). Unless otherwise provided, ligation may be accomplished usingknown buffers and conditions with 10 units to T4 DNA ligase ("ligase")per 0.5 μg of approximately equimolar amounts of the DNA fragments to beligated.

Unless otherwise stated, transformation was performed as described inthe method of Graham, F. and Van der Eb, A., Virology, 52:456-457(1973).

EXAMPLE 1

Bacterial Expression and Purification of Serum Paraoxonase

The DNA sequence encoding for paraoxonase, ATCC #75773, is initiallyamplified using PCR oligonucleotide primers corresponding to the 5' andsequences of the processed paraoxonase protein (minus the signal peptidesequence) and the vector sequences 3' to the paraoxonase gene.Additional nucleotides corresponding to paraoxonase were added to the 5'and 3' sequences respectively. The 5' oligonucleotide primer has thesequence 5' TCAGGATCCAGAAATCGACTTAAAGCCTCC 3' contains a Bam HIrestriction enzyme site followed by 21 nucleotides of paraoxonase codingsequence starting from the presumed terminal amino acid of the processedprotein codon. The 3' sequence 5' TCAAAGCTTTTAGAGTTCACAATACAAGGC 3'contains complementary sequences to a Hind III restriction site and isfollowed by 21 nucleotides of paraoxonase. The restriction enzyme sitescorrespond to the restriction enzyme sites on the bacterial expressionvector pQE-9 (Qiagen, Inc. 9259 Eton Avenue, Chatsworth, CA, 91311).pQE-9 encodes antibiotic resistance (Ampr), a bacterial origin ofreplication (ori), an IPTG-regulatable promoter operator (P/O), aribosome binding site (RBS), a 6-His tag and restriction enzyme sites.pQE-9 was then digested with Bam HI and Hind III. The amplifiedsequences were ligated into pQE-9 and were inserted in frame with thesequence encoding for the histidine tag and the RBS. The ligationmixture was then used to transform E. coli strain SURE (available fromStratagene Cloning Systems, 11099 North Torrey Pines Road, La Jolla,Calif. 92037) by the procedure described in Sambrook, J. et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press,(1989). Transformants are identified by their ability to grow on LBplates and ampicillin/kanamycin resistant colonies were selected.Plasmid DNA was isolated and confirmed by restriction analysis. Clonescontaining the desired constructs were grown overnight (O/N) in liquidculture in LB media supplemented with both Amp (100 ug/ml) and Kan (25ug/ml). The O/N culture is used to inoculate a large culture at a ratioof 1:100 to 1:250. The cells were grown to an optical density 600(O.D.⁶⁰⁰) of between 0.4 and 0.6. IPTG ("Isopropyl-B-D-thiogalactopyranoside") was then added to a final concentration of 1 mM. IPTGinduces by inactivating the lacI repressor, clearing the P/O leading toincreased gene expression. Cells were grown an extra 3 to 4 hours. Cellswere then harvested by centrifugation. The cell pellet was solubilizedin the chaotropic agent 6 Molar Guanidine HCl. After clarification,solubilized paraoxonase was purified from this solution bychromatography on a Nickel-Chelate column under conditions that allowfor tight binding by proteins containing the 6-His tag. Hochuli, E. etal., J. Chromatography 411:177-184 (1984). paraoxonase was eluted fromthe column in 6 molar guanidine HCl pH 5.0 and for the purpose ofrenaturation adjusted to 3 molar guanidine HCl, 100mM sodium phosphate,10 mmolar glutathione (reduced) and 2 mmolar glutathione (oxidized).After incubation in this solution for 12 hours the protein was dialyzedto 10 mmolar sodium phosphate.

EXAMPLE 2

Expression of Recombinant Paraoxonase in CHO cells

The expression of plasmid, paraoxonase HA is derived from a vectorpcDNAI/Amp (Invitrogen) containing: 1) SV40 origin of replication, 2)ampicillin resistance gene, 3) E.coli replication origin, 4) CMVpromoter followed by a polylinker region, a SV40 intron andpolyadenylation site. A DNA fragment encoding the entire paraoxonaseprecursor fused in frame to its 3' end was cloned into the polylinkerregion of the vector, therefore, the recombinant protein expression isdirected under the CMV promoter.

The plasmid construction strategy is described as follows:

The DNA sequence encoding for paraoxonase, ATCC #75773, was constructedby PCR on the original EST clone using two primers: the 5' primer 5'CGCGGGATCCACCATGGGGGCGGCTGGTGGCTCT 3' contains a Bam HI restriction sitefollowed by 21 nucleotides of paraoxonase coding sequence starting fromthe initiation codon; the 3' sequence 5'CGCGTCTAGACGCTTAGAGTTCACAATACAAGGC 3' contains complementary sequencesto an Xba I site and a translation stop codon and the last 18nucleotides of the paraoxonase coding sequence (not including the stopcodon). Therefore, the PCR product contains a Bam HI site, paraoxonasecoding sequence, a translation termination stop codon and an Xba I site.The PCR amplified DNA fragment and the vector, pcDNAI/Amp, were digestedwith Bam HI and Xba I restriction enzyme and ligated. The ligationmixture was transformed into E. coli strain SURE (available fromStratagene Cloning Systems, 11099 North Torrey Pines Road, La Jolla,Calif. 92037) the transformed culture was plated on ampicillin mediaplates and resistant colonies were selected. Plasmid DNA was isolatedfrom transformants and examined by restriction analysis for the presenceof the correct fragment. For expression of the recombinant paraoxonase,CHO cells were transfected with the expression vector by DEAE-DEXTRANmethod. (J. Sambrook, E. Fritsch, T. Maniatis, Molecular Cloning: ALaboratory Manual, Cold Spring Laboratory Press, (1989)). The expressionof the paraoxonase protein was detected by radiolabelling andimmunoprecipitation method. (E. Harlow, D. Lane, Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory Press, (1988)). Cellswere labelled for 8 hours with ³⁵ S-cysteine two days post transfection.Culture media were then collected and cells were lysed with detergent(RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50 mMTris, pH 7.5). (Wilson, I. et al., Id. 37:767 (1984)). Proteinsprecipitated were analyzed on 15% SDS-PAGE gels.

EXAMPLE 3

Expression pattern of paraoxonase in human tissue

Northern blot analysis is carried out to examine the levels ofexpression of paraoxonase in human tissues. Total cellular RNA samplesare isolated with RNAzol™ B system (Biotecx Laboratories, Inc. 6023South Loop East, Houston, Tex. 77033). About 10μg of total RNA isolatedfrom each human tissue specified is separated on 1% agarose gel andblotted onto a nylon filter. (Sambrook, Fritsch, and Maniatis, MolecularCloning, Cold Spring Harbor Press, (1989)). The labeling reaction isdone according to the Stratagene Prime-It kit with 50ng DNA fragment.The labeled DNA is purified with a Select-G-50 column. (5 Prime-3 Prime,Inc. 5603 Arapahoe Road, Boulder, Colo. 80303). The filter is thenhybridized with radioactive labeled full length paraoxonase gene at1,000,000 cpm/ml in 0.5M NaPO₄, pH 7.4 and 7% SDS overnight at 65° C.After wash twice at room temperature and twice at 60° C. with 0.5 ×SSC,0.1% SDS, the filter is then exposed at -70° C. overnight with anintensifying screen.

Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, within thescope of the appended claims, the invention may be practiced otherwisethan as particularly described.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 2                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1079 BASE PAIRS                                                   (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       CCCATGGGGCGGCTGGTGGCTGTGGGCTTGCTGGGGATCGCCCTGGCCCTCCTGGGCGAG60                AGGCTTCTGGCACTCAGAAATCGACTTAAAGCCTCCAGAGAAGTAGAATCTGTAGACCTT120               CCACACTGCCACCTGATTAAAGGAATTGAAGCTGGCTCTGAAGATATTGACATACTTCCC180               AATGGTCTGGCTTTTTTAAGTGTGGGTCTAAAATTCCCAGGACTCCACAGCTTTGCACCA240               GATAAGCCTGGAGGTATACTAATGATGGTTCTAAAAGAAGCAAAACCAAGGGGACGGGAA300               TTAAGAATCAGTCGTGGGTTTGATTTGGCCTCATTCAATCCACATGGGATCAGCACTTTC360               ATAGACAACGATGACACAGTTTATCTCTTGGTTGTAAACCACCCAGAATTCAAGAATACA420               GTGGAAATTTTTAATTTGGAAGAAGCAGAAAATTCTCTGTTGCATCTGAAAACAGTCAAA480               CATGAGCTTCTTCCAAGTGTGAATGACATCACAGCTGTTGGACCGGCACATTTCTATGCC540               ACAAATGACCACTACTTCTCTGATCCTTTCTTAAAGTATTTAGAAACATACTTGGAATTA600               CACTGGGCAAATGTTGTTTACTACAGGCCAAATGAAGTTAAAGGTGGTAGCAGGAAGGAT660               TTGGATTCAGCAAATGGGATCAATATTTCACCTGGATGGATAAGTTTTTCTATGTTGGCT720               GACATATTGGCTCATGAAATTCATGTTTGGGGAAAACACACTAATATGAATTTAACTCAG780               TTGAAGGTACTTGAGCTGGATACACTGGTGGATAATTTATCTATTGATCCTTCCTCGGGG840               GACATCTGGGTAGGCTGTCATCCTAATGGCCAGAAGCTCTTCGTGTATGACCCGAACAAT900               CCTCCCTCGTCAGAGGTTCTCCGCATCCAGAACATTCTATCTGAGAAGCCTACAGTGACT960               ACAGTTTATGCCAACAATGGGTCTGTTCTCCAAGGAAGTTCTGTAGGCTCAGTGTATGAT1020              GGGAAGCTGCTCATAGGCACTTTATACCACAGAGCCTTGTATTGTGAACTCTAAATTGT1079               (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 356 AMINO ACIDS                                                   (B) TYPE: AMINO ACID                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: PROTEIN                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetGlyArgLeuValAlaValGlyLeuLeuGlyIleAlaLeuAla                                 51015                                                                         LeuLeuGlyGluArgLeuLeuAlaLeuArgAsnArgLeuLysAla                                 202530                                                                        SerArgGluValGluSerValAspLeuProHisCysHisLeuIle                                 354045                                                                        LysGlyIleGluAlaGlySerGluAspIleAspIleLeuProAsn                                 505560                                                                        GlyLeuAlaPheLeuSerValGlyLeuLysPheProGlyLeuHis                                 657075                                                                        SerPheAlaProAspLysProGlyGlyIleLeuMetMetValLeu                                 808590                                                                        LysGluAlaLysProArgGlyArgGluLeuArgIleSerArgGly                                 95100105                                                                      PheAspLeuAlaSerPheAsnProHisGlyIleSerThrPheIle                                 110115120                                                                     AspAsnAspAspThrValTyrLeuLeuValValAsnHisProGlu                                 125130135                                                                     PheLysAsnThrValGluIlePheAsnLeuGluGluAlaGluAsn                                 140145150                                                                     SerLeuLeuHisLeuLysThrValLysHisGluLeuLeuProSer                                 155160165                                                                     ValAsnAspIleThrAlaValGlyProAlaHisPheTyrAlaThr                                 170175180                                                                     AsnAspHisTyrPheSerAspProPheLeuLysTyrLeuGluThr                                 185190195                                                                     TyrLeuGluLeuHisTrpAlaAsnValValTyrTyrArgProAsn                                 200205210                                                                     GluValLysGlyGlySerArgLysAspLeuAspSerAlaAsnGly                                 215220225                                                                     IleAsnIleSerProGlyTrpIleSerPheSerMetLeuAlaAsp                                 230235240                                                                     IleLeuAlaHisGluIleHisValTrpGlyLysHisThrAsnMet                                 245250255                                                                     AsnLeuThrGlnLeuLysValLeuGluLeuAspThrLeuValAsp                                 260265270                                                                     AsnLeuSerIleAspProSerSerGlyAspIleTrpValGlyCys                                 275280285                                                                     HisProAsnGlyGlnLysLeuPheValTyrAspProAsnAsnPro                                 290295300                                                                     ProSerSerGluValLeuArgIleGlnAsnIleLeuSerGluLys                                 305310315                                                                     ProThrValThrThrValTyrAlaAsnAsnGlySerValLeuGln                                 320325330                                                                     GlySerSerValGlySerValTyrAspGlyLysLeuLeuIleGly                                 335340345                                                                     ThrLeuTyrHisArgAlaLeuTyrCysGluLeu                                             350355                                                                        __________________________________________________________________________

What is claimed is:
 1. An isolated polynucleotide comprising apolynucleotide having at least a 95% identity to a member selected fromthe group consisting of:(a) a polynucleotide encoding a polypeptidecomprising an amino acid sequence as set forth in SEQ ID NO:2; (b) apolynucleotide encoding a polypeptide comprising amino acid 25 to aminoacid 356 as set forth in SEQ ID NO:2; (c) a polynucleotide which iscomplementary to the polynucleotide of (a); and (d) a polynucleotidewhich is complementary to the polynucleotide of (b).
 2. Thepolynucleotide of claim 1 wherein the polynucleotide is DNA.
 3. Thepolynucleotide of claim 1 wherein the polynucleotide is RNA.
 4. Theisolated polynucleotide of claim 1 wherein said polynucleotide encodes apolypeptide comprising an amino acid sequence set forth in SEQ ID NO:2.5. The isolated polynucleotide of claim 4 wherein said polynucleotide isDNA.
 6. The isolated polynucleotide of claim 1 wherein saidpolynucleotide encodes a polypeptide comprising amino acid 25 to aminoacid 356 of SEQ ID NO:2.
 7. The isolated polynucleotide of claim 6wherein said polynucleotide is DNA.
 8. The polynucleotide of claim 2wherein said polynucleotide comprises nucleotide 4 to nucleotide 1071 ofSEQ D NO:1.
 9. The polynucleotide of claim 2 wherein said polynucleotidecomprises nucleotides 78 to nucleotide 1079 of SEQ ID NO:1.
 10. Anisolated polynucleotide comprising polynucleotide having at least a 95%identity to a member selected from the group consisting of:(a) apolynucleotide encoding a polypeptide encoded by human cDNA contained inATCC Deposit No. 75773; (b) a polynucleotide encoding a maturepolypeptide encoded by human cDNA contained in ATCC Deposit No. 75773;(c) a polynucleotide which is complementary to the polynucleotide of(a); and (d) a polynucleotide which is complementary to thepolynucleotide of (b).
 11. The isolated polynucleotide of claim 10wherein said polynucleotide encodes a polypeptide expressed by the humancDNA contained in ATCC Deposit No.
 75773. 12. The isolatedpolynucleotide of claim 10 wherein said polynucleotide encodes a maturepolypeptide encoded by the human cDNA contained in ATCC Deposit No.75773.
 13. A vector containing the polynucleotide of claim
 2. 14. Avector containing the polynucleotide of claim
 4. 15. A vector containingthe polynucleotide of claim
 5. 16. A vector containing thepolynucleotide of claim
 6. 17. A vector containing the polynucleotide ofclaim
 7. 18. A vector containing the polynucleotide of claim
 8. 19. Avector containing the polynucleotide of claim
 9. 20. A vector containingthe polynucleotide of claim
 11. 21. A vector containing thepolynucleotide of claim
 12. 22. A host cell transformed or transfectedwith the vector of claim
 13. 23. A host cell transformed or transfectedwith the vector of claim
 14. 24. A host cell transformed or transfectedwith the vector of claim
 15. 25. A host cell transformed or transfectedwith the vector of claim
 16. 26. A host cell transformed or transfectedwith the vector of claim
 17. 27. A host cell transformed or transfectedwith the vector of claim
 18. 28. A host cell transformed or transfectedwith the vector of claim
 19. 29. A host cell transformed or transfectedwith the vector of claim
 20. 30. A host cell transformed or transfectedwith the vector of claim 21.